Gene expression in mammalian cells

ABSTRACT

Proteins such as human β-interferon or human erythropoietin are prepared by culturing mammalian cells which harbour a nucleic acid sequence comprising: (i) a coding sequence which encodes the desired protein and which is operably linked to a promoter capable of directing expression of the coding sequence in a mammalian cell in the presence of a heavy metal ion; and (ii) a first selectable marker sequence comprises a metallothionein gene and which is operably linked to a promoter capable of directing expression of the metallothionein gene in a mammalian cell in the presence of a heavy metal ion; and optionally (iii) a second selectable marker sequence which comprises a neo gene and which is operably linked to a promoter capable of directing expression of the neo gene in a mammalian cell.

This invention relates to the expression of genes in mammalian cells,particularly genes responsible for proteins whose biological activity invivo is affected by a diversity of factors including specificglycosylation. Examples of such genes are the human β-interferon (IFNβ),human erythropoietin (EPO), human chorionic gonadotropin, various othercytokines and growth factors as well as specific viral antigens such asDengue viral proteins whose structure may be relevant for thedevelopment of vaccines.

Previously, genes have been extensively expressed in mammalian celllines, particularly in mutant Chinese Hamster Ovary (CHO) cellsdeficient in the dihydrofolate reductase gene (dhfr) as devised by themethod of Urlaub et al, PNAS U.S.A. 77, 4216-4220, 1980. A variety ofexpression systems have been used. Many vectors for the expression ofgenes in such cells are therefore available. Typically, the selectionprocedures used to isolate cells transformed with the expression vectorsrely on using methotrexate to select for transformants in which both thedhfr and the target genes are coamplified.

The dhfr gene, which enables cells to withstand methotrexate, is usuallyincorporated in the vector with the gene whose expression is desired.Selection of cells under increasing concentrations of methotrexate isthen performed. This leads to amplification of the number of dhfr genespresent in each cell of the population, as cells with higher copynumbers withstand greater concentrations of methotrexate. As the dhfrgene is amplified, the copy number of the gene of interest increasesconcomitantly with the copy number of the dhfr gene, so that increasedexpression of the gene of interest is achieved.

Unfortunately, these amplified genes have been reported to be variablyunstable in the absence of continued selection (Schimke, J. Biol. Chem.263, 5989-5992, 1988). This instability is inherent to the presentlyavailable expression systems of CHO dhfr⁻ cells.

For many years, several promoters have been used to drive the expressionof the target genes such as the SV40 early promoter, the CMV earlypromoter and the SRα promoter. The CMV and SRα promoters are claimed tobe the strongest (Wenger et al, Anal. Biochem. 221, 416-418, 1994).

In one report, the β-interferon promoter has also been used to drive theexpression of the β-interferon gene in the mutant CHO dhfr⁻ cells (U.S.Pat. No. 5,376,567). In this system, however, the selected CHO dhfr⁻cells had to be superinduced by the method of Tan et al (Tan et al, PNASU.S.A. 67, 464-471, 1970; Tan et al, U.S. Pat. No. 3,773,924) to effecta higher level of β-interferon production. In this system a significantpercentage of the superinduced β-interferon produced by the CHO dhfr⁻cells was not glycosylated.

The mouse metallothionein gene (mMT1) promoter has also been used forthe expression of β-interferon genes in CHO cells, BHK and LTK⁻ mousecells (Reiser et al 1987 Drug Res. 37, 4, 482-485). However, theexpression of β-interferon with this promoter was not as good as theSV40 early promoter in CHO cells. Further, β-interferon expression fromthese cells mediated by the mMT1 promoter was inducible by heavy metals.Heavy metals are however extremely toxic to the cells and this systemwas therefore abandoned. Instead, Reiser et al used the CHO dhfr⁻expression system in conjunction with the SV40 early promoter (Reiser etal, Drug Res. 37,4, 482-485 (1987) and EP-A-0529300) to produceβ-interferon in CHO dhfr⁻ cells as derived by the method of Urlaub et al(1980).

We have now expressed β-interferon in wild-type CHO cells. Wild-type CHOcells were transfected with a vector comprising a β-interferon geneunder the control of a mouse sarcoma viral enhancer and mousemetallothionein promoter (MSV-mMT1), a neo gene under the control ofpromoter capable of driving expression of the neo gene in both E. coliand mammalian cells and a human metallothionein gene having its ownpromoter. Transfected cells capable of expressing β-interferon wereselected by first exposing cells to geneticin (antiobiotic G418) andthus eliminating cells lacking the neo gene and then exposing thesurviving cells to increasing concentrations of a heavy metal ion.

The heavy metal ion enhanced the MSV-mMT1 promoter for the β-interferongene, thus increasing β-interferon expression. The heavy metal ion alsoinduced the human metallothionein gene promoter, causing expression ofhuman metallothionein. The human metallothionein protected the cellsagainst the toxic effect of the heavy metal ion. The presence of theheavy metal ion ensured that there was continual selection of cellswhich had the transfecting vector, or at least the β-interferon gene andthe human metallothionein gene and their respective promoters,integrated into their genome.

The selected cells that had been successfully transfected expressedβ-interferon. Expression was surprisingly improved when the cells werecultured in the presence of Zn²⁺. The β-interferon had improvedproperties, in particular a higher bioavailability, than priorβ-interferons.

These findings have general applicability. Accordingly, the presentinvention provides:

a nucleic acid vector comprising:

(i) a coding sequence which encodes a protein of interest and which isoperably linked to a promoter capable of directing expression of thecoding sequence in a mammalian cell in the presence of a heavy metalion; (ii) a first selectable marker sequence which comprises ametallothionein gene and which is operably linked to a promoter capableof directing expression of the metallothionein gene in a mammalian cellin the presence of a heavy metal ion; and (iii) a second selectablemarker sequence which comprises a neo gene and which is operably linkedto a promoter capable of directing expression of the neo gene in amammalian cell;

mammalian cells which harbour a nucleic acid sequence comprising:

(i) a coding sequence which encodes a protein of interest and which isoperably linked to a promoter capable of directing expression of thecoding sequence in a mammalian cell in the presence of a heavy metalion; (ii) a first selectable marker sequence which comprises ametallothionein gene and which is operably linked to a promoter capableof directing expression of the metallothionein gene in a mammalian cellin the presence of a heavy metal ion; and optionally (iii) a secondselectable marker sequence which comprises a neo gene and which isoperably linked to a promoter capable of directing expression of the neogene in a mammalian cell;

a process for producing such cells, which process comprises

(a) transfecting mammalian cells with a vector of the invention; (b)exposing the transfected cells to geneticin to eliminate thereby cellslacking the neo gene; and (c) exposing the cells that survive step (a)to progressively increasing concentrations of a heavy metal ion toselect thereby the desired cells.

use of a neo gene and a metallothionein gene as selectable marker genesin a single vector; and a process for the preparation of a protein ofinterest, which process comprising culturing mammalian cells of theinvention under conditions allowing expression of the desired proteinand recovering the desired protein thus expressed.

By using both a neo gene and a metallothionein gene as selectablemarkers in a single vector, it is possible to select for transformedmammalian cells, such as wild-type CHO cells, which have multiple copiesof the expression vector stably integrated into their genomes. Thisselection system therefore facilitates the preparation andidentification of stably transformed mammalian cells such as thewild-type CHO cells and avoids the need for dhfr⁻ cells. The transformedcells enable the stable expression of genes such as the humanβ-interferon gene because they have multiple copies, typically at least20-100 copies or more, of these genes integrated into their genomes.

Moreover, the use of relatively high concentrations of Cd²⁺ (up to 200μM) in the selection procedure eliminates inadvertent microbialcontaminants such as mycoplasma that may become associated with thetransfected cells during tissue culture procedures. Thus, the presentinvention minimises the possibility of microbial contamination oftransfected cells.

Further, one particular promoter/enhancer system according to theinvention surprisingly gave a significantly higher level of expressionthan the strong promoter systems that have been used in the past. Thispromoter/enhancer is the MSV-mMT1 system which comprises the promoter ofthe mouse metallothionein gene 1 (mMT1) flanked upstream with a mousesarcoma virus (MSV) enhancer.

A promoter of a metallothionein gene, particularly the combined MSV-mMT1promoter/enhancer system, can be operably linked to a gene of interestsuch as the human β-interferon gene or the human erythropoietin gene. Avector comprising such an arrangement can give a high level ofexpression of the gene product in wild-type CHO cells. Therefore, theinventors have identified a new and unexpectedly powerful expressionsystem suitable for use in mammalian cells, particularly wild typemammalian cells. Products, such as human β-interferon and humanerythropoietin, may be expressed with unexpected/novel biologicalproperties such as higher bio-availability. Such properties may resultin higher efficacy/additional utility for the product.

It is therefore possible according to the invention to express genessuch as a β-interferon gene and others in large quantities in wild-typemammalian cells such as wild-type CHO cells and to do so in a stablemanner, without the need for continuing selection and dependence on theCHO dhfr⁻-methotrexate selection system. The invention can be applied toa large variety of mammalian cells. In this way, it enables theexpression of appropriate target genes with a glycosylation pattern anda cellular environment unique to the cell type used.

A vector according to the invention is an expression vector. Itcomprises three sequences that are expressible in mammalian cells. Thusa vector of the invention comprises:

(i) a coding sequence comprising a gene of interest whose expression isdesired, for example the human β-interferon gene; (ii) a firstselectable marker sequence comprising a metallothionein gene whichconfers resistance to heavy metal ions, such as cadmium, copper andzinc, on mammalian cells expressing the gene of interest; and (iii) asecond selectable marker sequence comprising a neo gene which confersresistance to the antibiotic kanamycin upon transformed bacterial cellsexpressing the gene and resistance to the geneticin (antibiotic G418)upon mammalian cells expressing the gene.

Each of these three sequences will typically be associated with otherelements that control their expression. In relation to each sequence,the following elements are generally present, usually in a 5′ to 3′arrangement: a promoter for directing expression of the sequence andoptionally a regulator of the promoter, a translational start codon, thecoding/marker sequence, a polyadenylation signal and a transcriptionalterminator. Further, the coding sequence and/or either or both of themarker sequences may optionally be operably linked to an enhancer thatincreases the expression obtained under the control of the promoter.Suitable enhancers include the Rous Sarcoma Virus (RSV) enhancer and theMouse Sarcoma Virus (MSV) enhancer.

Further, a vector according to the invention will typically comprise oneor more origins of replication, for example a bacterial origin ofreplication, such as the pBR322 origin, that allows replication inbacterial cells. Alternatively or additionally, one or more eukaryoticorigins of replication may be included in the vector so that replicationis possible in, for example yeast cells and/or mammalian cells.

The vector may also comprise one or more introns or other non-codingsequences 3′ or 5′ to the coding sequence or to one or more of themarker sequences. Such non-coding sequences may be derived from anyorganism, or may be synthetic in nature. Thus, they may have anysequence. Such sequences may be included if they enhance or do notimpair correct expression of the coding sequence or marker sequences.

In vectors of the invention, the coding sequence and the markersequences are each operably linked to a promoter capable of directingtheir expression in a mammalian cell. Optionally, one or more of thesepromoters may also be capable of directing expression in other cells,for example non-mammalian eukaryotic cells, such as yeast cells orinsect cells and/or prokaryotic cells. “Operably linked” refers to ajuxtaposition wherein the promoter and the coding/marker sequence are ina relationship permitting the coding/marker sequence to be expressedunder the control of the promoter. Thus, there may be elements such as5′ non-coding sequence between the promoter and coding/marker sequence.Such sequences can be included in the construct if they enhance or donot impair the correct control of the coding/marker sequence by thepromoter.

Any promoter capable of enhancing expression in a mammalian cell in thepresence of a heavy metal ion such as Cd²⁻, Cu²⁻ and Zn²⁺ may beoperably linked to the coding sequence. A suitable promoter is ametallothionein gene promoter. The mouse metallothionein gene I (MMT1)promoter is preferred.

Suitable promoter/enhancer combinations for the encoding sequenceinclude the mTM1 promoter flanked upstream with MSV enhancer (MSV-mMT1)and the combination of the RSV enhancer and the MMTV promoter. MSV-mMT1is preferred.

Similarly, any promoter capable of enhancing expression in a mammaliancell in the presence of a heavy metal ion such as Cd²⁺, Cu²⁺ and Zn²⁺may be operably linked to the metallothionein gene such as a humanmetallothionein gene. Preferably, the marker sequence gene is a humanmetallothionein gene, such as the human metallothionein gene IIA, whichhas its own promoter.

The second selectable marker sequence is a neo gene. More than one typeof this gene exists in nature: any specific neo gene can be used in avector of the invention. One preferred neo gene is the E. coli neo gene.

The promoter for the neo gene is capable of directing expression of thegene in a mammalian cell. Suitable promoters are the cytomegalovirus(CMV) early promoter, the SV40 promoter, the mouse mammary tumour viruspromoter, the human elongation factor 1 α-P promoter (EF-1α-P), the SRαpromoter and a metallothionein gene promoter such as mMT1. The promotermay also be capable of expressing the neo gene in bacteria such as E.coli in which a vector of the invention may be constructed.

Whilst the protection against antibiotics conferred by the neo gene isqualitative in the sense that once expressed neo gene will conferantibiotic resistance on a cell, the protection against heavy metalsconferred by the metallothionein gene is quantitative. The greater thelevel of expression of the metallothionein gene in a cell the greaterthe cell's resistance is to heavy metals. Thus, cells having a high copynumber of metallothionein genes will be expected to have a highresistance to heavy metals.

Therefore, cells including many copies of a vector of the invention havea higher resistance to heavy metals than cells comprising one or a fewcopies. Accordingly, it is possible to select for transfected cellshaving high copy numbers of a vector of the invention (and thereforehigh copy numbers of the coding sequence for a gene such as humanβ-interferon) by progressively increasing the concentration of heavymetals to which the cells are exposed. Thus, cells having progressivelyhigher copy numbers of the vector according to the invention areselected.

Therefore, the combination of selectable markers found in the vectors ofthe invention allows a two stage selection process for transfected cellsof interest. First, cells are exposed to geneticin (antiobiotic G418)which eliminates cells lacking the neo gene and therefore lacking thevector of the invention altogether. The neo gene serves no furtherfunction after this step.

Second, selection is effected with progressively increasing levels ofheavy metal ions, which selects cells having multiple copies of thevectors, especially cells having multiple copies integrated into theirgenomes. In this selection process, cells that survive highconcentrations of heavy metal ions express metallothionein to a highdegree, for example because they include a large number of vectors ofthe invention and/or because the vector or vectors that have integratedinto their genome are in a chromosomal location that encourages strongexpression.

Any suitable heavy metal ions may be used. Thus, any heavy metal ionthat is toxic to cells of the invention and to which an expressedmetallothionein gene confers protection may be used. For example, zincions (Zn²⁻), copper ions (Cu²⁻) or preferably cadmium ions (Cd²⁺) may beused. Concentrations of a heavy metal ion of from 5 to 100, indeed up to200, μM may be applied to effect selection. A concentration of from 130to 170 μM, preferably about 150 μM Zn²⁻, is suitable.

In order to effect selection using heavy metal ions, these ions may beprovided as salts, in combination with any suitable counterion such assulphate or chloride.

Because selected cells are resistant to the toxicity of heavy metalswhich, as it happens, are inducers of the promoter for the codingsequence, the expression of the protein of interest can be maximised byheavy metal ions such as 130 to 170 μM Zn²⁻ which are inducers of thepromoter.

In addition to the neo and metallothionein genes, the vector may alsocontain one or more further selectable marker genes, for example anampicillin resistance gene for the identification of bacterialtransformants.

In the vectors of the invention, the nucleic acid may be DNA or RNA,preferably DNA. The vectors may be expression vectors of any type. Thevector must of course be compatible with the mammalian cell which it isgoing to transfect. The vector may be in linear or circular form. Forexample, the vector may be a plasmid vector, typically a DNA plasmid. Apreferred plasmid vector is pMMTC (Example 2; FIG. 3).

Those of skill in the art will be able to prepare suitable vectorsstarting with widely available vectors which will be modified by geneticengineering techniques such as those described by Sambrook et al(Molecular Cloning: A Laboratory Manual: 1999). So far as plasmidvectors are concerned, a suitable starting vector is the plasmid pRSN(Low et al (1991): JBC 266; 19710-19716), which is widely available. Afurther suitable plasmid starting vector is pBR322.

Vectors of the invention may be able to effect integration of some orall of their nucleic acid sequence into a host cell genome or they mayremain free in the host cell. Integrative vectors are preferred. This isbecause they give stable expression of coding sequences such as that ofthe human β-interferon gene.

The transfected mammalian cells may be BHK, COS, VERO, humanfibroblastoid such as ClO, HeLa, or human lymphoblastoid cells or cellsof a human tumour cell line. Preferably, however, the cells are CHOcells, particularly wild-type CHO cells.

Desirably, transfected cells will have all or part of a vector of theinvention integrated into their genomes. Such cells are preferredbecause they give stable expression of the coding sequence contained inthe vector. Preferably, one or more copies of the entire vector will beintegrated, with cells having multiple integrated copies of the vector,for example from 20 to 100 copies or more, being particularly preferredbecause these cells give a high stable level of expression of the codingsequence contained in the vector. However, cells having less thancomplete sections of vectors of the invention integrated into theirgenomes are also included within the invention if they are functionallyequivalent to cells having the entire vector integrated into theirgenomes, in the sense that the integrated sections of the vector enablethe cell to express the coding sequence and to be selected for by theuse of heavy metals, as described above. Thus, cells exhibiting partialintegration of vector of the invention are included in the invention ifthe integrated element or elements include the coding sequence operablylinked to its associated promoter and the metallothionein markersequence operably linked to its associated promoter.

The cells may be transfected by any suitable method, such as the methodsdisclosed by Sambrook et al (Molecular cloning: A Laboratory Manual,1989). For example, vectors comprising nucleic acid sequences accordingto the invention may be packaged into infectious viral particles, suchas retroviral particles. The vectors may also be introduced byelectroporation, calcium phosphate precipitation or by contacting nakednucleic acid vectors with the cells in solution. Preferred methods oftransfection include those described by Low et al (JBC 266; 19710-19716;1991).

The invention also provides a process for producing proteins encoded bythe coding sequence in a vector of the invention. Such processescomprise culturing cells transfected with a vector of the inventionunder conditions that allow expression of the coding sequence andrecovering the thus produced protein. Preferred proteins that may beproduced in this way include interferons, for example human interferons.β-interferons are preferred and human β-interferon is most preferred.Other proteins are interleukins (such as interleukin-12), humanchorionic gonadotropin, growth factors, human growth hormone and humanerythropoietin, cell membrane components, viral proteins and otherproteins of biomedical relevance.

The selected cells may be cultured under any suitable conditions knownin the art and these conditions may vary depending on the cell type andthe type of protein being produced. The promoter for the coding sequencecan be a constitutive promoter so that the protein encoded by the codingsequence is expressed in the absence of a heavy metal ion. The cells mayhowever be cultured in the presence of a heavy metal ion, particularlyin an amount which is not toxic to the cells. That can lead to higherexpression of the desired protein.

The concentration of the heavy metal ion in the culture medium istypically from 100 to 200 μM. Cells may therefore be cultured in thepresence of from 100 to 200 μM of a heavy metal ion selected from Cd²⁺,Cu²⁺ and Zn²⁺. for example from 130 to 170 μM of the heavy metal ion. Auseful concentration is about 150 μM, particularly when the heavy metalion is Zn²⁺. The use of Zn²⁺ has a beneficial effect on the yield ofβ-interferon and erythropoietin production. Unexpectedly, it wasobserved that human β-interferon production was increased two- tothree-fold and human erythropoietin production was increased three- tofive-fold.

The protein that is produced may be recovered by any suitable meansknown in the art and the method of recovery may vary depending on thetype of cells employed, the culture conditions and the type of proteinbeing produced. Desirably, the protein produced will be purified afterrecovery. Substantially pure protein can thus be obtained.

The present invention enables a novel human β-interferon to be provided.This β-interferon has a high degree of sialylation. Like natural humanβ-interferon produced by primary diploid human fibroblasts, it is wellglycosylated. However, it has a higher bioavailability than the naturalβ-interferon or recombinant β-interferon produced in E. coli(BETASERON).

The higher bioavailability of the β-interferon can be characterised.When 1.5×10⁶ International Units (I.U.) of the interferon is injectedsubcutaneously into the back of a rabbit of about 2 kg: (a) ≧128 I.U./mlof the interferon is detectable in the serum of the rabbit after 1 hour,and/or (b) ≧64 I.U./ml of the interferon is detectable in the serum ofthe rabbit after 5 hours.

The maximum level of interferon is typically observed; after 1 hour.According to (a), therefore, 128 to 256 I.U./ml such as 140 to 190I.U./ml of the interferon may be detectable in the rabbit serum after 1hour. After 5 hours according to (b), ≧70 I.U./ml such as ≧80 I.U./ml ofthe interferon may be detectable in the rabbit serum. Typicallyaccording to (b), an amount of interferon in the range of 64 to 128I.U./ml such as 80 to 110 I.U./ml can be detected.

Additionally or alternatively, the interferon can be characterised byits specific activity. It can have a specific activity in the range offrom 4.8×10⁸ to 6.4×10⁸ I.U. per mg equivalent of bovine serum albuminprotein. The specific activity may be from 5×10⁸ to 6×10⁸, for examplefrom 5.2×10⁸ to 5.8×10⁸ such as from 5.3×10⁸ to 5.5×10⁸, I.U. per mgequivalent of bovine serum albumin protein.

The specific activity can be referenced to a standard, in particular theGb23-902-531 standard distributed by the Natl. Inst. Allergy andInfectious Disease, NIH, U.S.A. Specific activity is determinedaccording to a modification of the method of Armstrong (1971) in which0.2 μg/ml of actinomycin D is included in the viral challenge and theviral-induced C.P.E. is read directly. The assay cells were MRC-5fibroblasts.

The β-interferon may also be characterised by one or more of thefollowing properties:

1. The β-interferon according to the present invention typically has anapparent molecular weight of 26,300 as determined by 15% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

2. When injected as a neat intravenous bolus into a rabbit, the halflife of the interferon is typically in the range of from 12 to 15 minsuch as about 13½ min. The bolus is injected into the rabbit ear veinand blood samples are withdrawn from the rabbit ear artery. Rabbit serumis assayed for the antiviral activity of the interferon according to themodification of the method of Armstrong (1971).

3. The antiviral activity of the interferon in a human hepatoblastomacell line (HepG2) is at least equal to and, typically, about 1.5 timesthe activity of natural β-interferon from primary diploid humanfibroblast cells. The interferon is also about 2.2 times more effectivethan betaseron in protecting Hep2 cells against a viral challenge.Antiviral activity is again determined according to the modified methodof Armstrong (1971). Actinomycin D was omitted in the antiviraldetermination in HepG2 cells. The oligosaccharides associated with theβ-interferon of the invention may also characterise the β-interferon.The β-interferon carries oligosaccharides which can be characterised byone or more of the following features:

1. Neutral (no acidic substituents): 5 to 15%, preferably about 10%.Acidic: 95 to 85%, preferably about 90%

2. The total desialylated oligosaccharide pool is heterogeneous with atleast six distinct structural components present in the pool.

3. Matrix-Assisted Laser Desorption Ionisation—Time of Flight(MALDI-TOF) mass spectrometry and high resolution gel permeationchromatography data are summarised as follows:

Mass Compo- Calculated gu detected sition Mass equivalent 1786.2 5Hex,1782 11.1 4HexNAc, 1 2AB, Na 1929.9 5Hex, 1dHex, 1928 12.2 4HexNAc, 12AB, Na 2295.5 6Hex, 2293 14.5 1dHex, 5HexNAc 1 2AB, Na 2660.1 7Hex,2658 17.6 1dHex, 6HexNAc 1 2AB, Na 3019.1 8Hex, 3023 20.7 1dHex,7HexNAc, 1 2AB, Na

The carbohydrate moiety of the β-interferon of the invention consists ofbi-, tri- and tetra-antennary complex type N-linked oligosaccharides.These oligosaccharides contain repeating lactosamine(s). About 30 to80%, for example 35 to 60% or 35 to 50%, of the oligosaccharides arebi-antennary oligosaccharides. About 15 to 65%, for example from 25 to50% or 25 to 40%, of the oligosaccharides are tri-antennaryoligosaccharides. About 5 to 55%, for example from IS to 45% or 20 to40%, of the oligosaccharides are tetra-antennary oligosaccharides.

The β-interferon of the invention exhibits antiviral activity, cellgrowth regulatory activity and an ability to regulate the production ofintracellular enzymes and other cell-produced substance. Accordingly,the β-interferon may be used to treat various viral and oncologicdiseases such as hepatitis B, hepatitis C, viral encephalitis, viralpneumonia, viral warts, AIDS/nasopharyngeal carcinoma, lung cancer,melanomas,CML renal cell carcinoma and brain tumours as well as diseaseslike multiple sclerosis, hemangiomas and cervical intraepithelialneoplasia.

Pharmaceutical compositions that contain the β-interferon of theinvention as an active principal will normally be formulated with anappropriate pharmaceutically acceptable carrier or diluent dependingupon the particular mode of administration being used. For instance,parenteral formulations are usually injectable fluids that usepharmaceutically and physiologically acceptable fluids such asphysiological saline, balanced salt solutions, or the like as a vehicle.Oral formulations, on the other hand, may be solids, e.g. tablets orcapsules, or liquid solutions or suspensions. The interferon of theinvention will usually be formulated as a unit dosage form that containsfrom 10⁴ to 10⁹, more usually 10⁶ to 10⁷, I.U. per dose.

The interferon may be administered to humans in various manners such asorally, intravenously, intramuscularly, intraperitoneally, intranasally,intradermally, and subcutaneously. The particular mode of administrationand dosage regimen will be selected by the attending physician takinginto account the particulars of the patient, the disease and the diseasestate involved. For instance, viral infections are usually treated bydaily or twice daily doses over a few days to a few weeks; whereas tumoror cancer treatment involves daily or multidaily doses over months oryears.

The interferon may be combined with other treatments and may be combinedwith or used in association with other chemotherapeutic orchemopreventive agents for providing therapy against viral infections,neoplasms, or other conditions against which it is effective. Forinstance, in the case of herpes virus keratitis treatment, therapy withinterferon has been supplemented by thermocautery, debridement andtrifluorothymidine therapy.

The following Examples illustrate the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of immunoblot analysis carried out on CHO cellstransfected with DPPIV sequences under the control of various promoters;

FIGS. 2 to 4 are vector maps of plasmids pMMTN, pMMTC and pBPVrespectively. The multiple cloning site (MCS) is SEQ ID NO:7.

FIG. 5(a) shows the results of SDS-PAGE (15%) analysis of purifiedGS38-IFNβ. The molecular weight markers are from BioRad (California).The molecular weight of the markers are indicated in kda. The amount ofGS38-IFNβ in lane 1, 2 and 3 are 0.8×10⁶ I.U., 0.2×10⁶ I.U. and 1.1×10⁶I.U. respectively. The amount of bovine serum albumin in lanes a, b, c,d, e and f are 5 μg, 3.5 μg, 2.5 μg, 1.5 μg, 0.5 μg, 0.25 μg.

FIG. 5(b) shows the results of a Western Blot of purified GS38-IFNβ.Purified IFNβ produced by primary human diploid fibroblasts (lanes 1 and2) and GS38-IFNβ (lanes 3 and 4) were subjected to SDS-PAGE (15%). Theproteins were then blotted onto nitrocellulose membrane and probed withan anti-IFNβ monoclonal antibody (Accurate Chem, New York.) The amountsof IFNβ activity in the various lanes are 0.1×10⁶, 0.05×10⁶, 0.14×10⁶and 0.56×10⁶ I.U. in lanes 1, 2, 3 and 4. The molecular weights in kdaare indicated in the figure.

FIG. 6 is an hplc anion exchange chromatogram of the oligosaccharidesassociated with GS38-IFNβ;

FIG. 7 is a hplc anion exchange chromatogram of the oligosaccharidesassociated with GS38-IFNβ after neurammidase treatment;

FIG. 8 is a high resolution gel permeation chromatogram of theoligosaccharides associated with GS38-IFNβ;

FIG. 9 shows the molecular weight distribution of the de-acidifiedglycans released from GS38-IFNβ by MALDI-TOF mass spectrometry.

FIG. 10(a) shows the serum levels of IFNβ in rabbits after subcutaneousinjection with 0.7×10⁶ I.U. of IFNβ produced by GS38 cells (♦), primaryhuman diploid fibroblasts (▪) or E. coli [BETASERON (▴)]. The weight ofrabbits injected was about 1.5 kg.

FIG. 10(b) shows the serum levels of IFNβ in rabbits after subcutaneousinjection with 1.5×10⁶ I.U. of IFNβ produced by GS38 cells (♦), primaryhuman diploid fibroblasts (▪) or E. coli [BETASERON (▴)]. The weight ofrabbits injected was about 2.0 kg.

FIG. 11 shows the decay of serum IFNβ levels in rabbits (about 2 kg)injected intravenously with 0.7×10⁶ I.U. of three different kinds ofIFNβ prepared from GS38 cells (♦), primary human diploid fibroblasts (▪)or E. coli [BETASERON (▴)].

FIG. 12 shows the dosage effect of subcutaneously injected GS38-IFNβ onserum levels of circulating GS38/IFNβ. Levels of serum GS38-producedIFNβ levels in rabbits (2 kg) injected subcutaneously with [1.2×10⁶I.U., (♦), 2.5×10⁶ I.U. (▪) and 10.1×10⁶ I.U. (▴) of GS38 IFNβ.

FIG. 13 concerns expression of human erythropoietin from wild type CHOcells transfected by the new vector containing the EPO instead of theIFNβ gene. Cells of the indicated clones (lane 1 to 14) were seeded onto35 mm (in diameter) culture dishes. Upon confluency, 1 ml of culturemedium was added to each of them and cultured for 24 hrs. The media wereharvested and 10 μl of each, together with a control 50 ng of humanerythropoietin (lane 15) were resolved by SDS-PAGE and analyzed byWestern blot using the Amersham ECL detection system.

EXAMPLE 1

Identification of MSV-mMT1 as a powerful promoter for wild-type CHOcells

The strengths of 5 promoter/enhancer systems were compared. These were:

mouse metallothionein gene 1 promoter flanked upstream with mousesarcoma virus enhancer (MSV-mMT1: mMT1 is described by Glanville et al(1981) Nature 292, 267-269; MSV is described by Dhar et al (1980) PNAS77, 3937-3941);

the cytomegalovirus early promoter (CMV);

RSV-SV40 (a fusion between the rous sarcoma virus [RSV] enhancer andSV40 early promoter);

RSV-mouse mammary tumour virus long terminal enhancer/promoter(RSV-MMTV); and

SR-α promoter (Yutaka Takebe et al (1988) Mol. Cell. Biol. 8, 466-472).

For this comparison, an EcoRI-XhoI cDNA encoding full-length dipeptidylpeptidase IV (DPPIV) (Hong and Doyle (1988) J. Biol. Chem. 263,16892-16898) was cloned into the respective expression vectors so thatDPPIV expression was under the control of MSV-mMT1, CMV, RSV-SV40,RSV-MMTV, or SR-α, respectively.

For MSV-mMT1, the DPPIV fragment was inserted into XhoI-NotI sites ofpMMTN vector (FIG. 2); for CMV, the fragment was inserted into the EcoRIand XhoI sites of pXJ41neo vector (Zheng and Pallen (1992); Nature,359,336-339); for RSV-SV40, see Low et al (1991) J. Biol. Chem. 266,19710-19716); for RSV-MATV, the fragment was inserted into the NhoI-XhoIsites of pMAMneo vector (from Clontech: catalogue number 6104-1;described by Lee et al (1981): Nature 294, 228 and by Sardet et al(1989): J. cell 56, 271); for SR-α, the fragment was inserted into theXhoI-BamHI sites of pSRalpha/neo vector (Yutaka Takebe et al (1988) MCB8, 466-472; Nilsson et al, J. Cell Biol. 120, 5-13, 1993).

Each expression vector was transfected into CHO cells and stablytransfected cells were pooled for each vector. The strength of eachexpression vector was then measured by the protein levels of DPPIV usingimmunoblot analysis, with the amount of DPPIV detected giving anindication of the strength of each expression system.

Immunoblot analysis to detect DPPIV in these transfected cells wasperformed as described by Hong et al (1989) (Biochemistry 28,8474-8479). Briefly, cells were washed with Tris-buffered saline (TBS)(20 mM Tris, pH7.2, 150 mM NaCl), and then extracted with 1% TRITONX-100 in TBS with 1 mM PMSF. The extracts were cleared of cell debris bycentrifugation. The protein concentration of the extracts was determinedusing a BSA kit (Pierce Chemical Co.).

About 100 μg of proteins extracted from respective transfected cellswere resolved by SDS-PAGE and then analysed by immunoblot as previously(Hong et al (1989) Biochemistry 28, 8474-8479). The results shown inFIG. 1 show that the MSV-mMT1 expression system (lane 2) is muchstronger than the remaining widely used ones.

EXAMPLE 2

Description of plasmid pMMTC

Based on the above, a powerful expression vector pMMTC was constructedusing MSV-mMT1 to drive the expression of foreign genes in conjunctionwith two selection markers (the neo gene for transfected cells and thehuman metallothionein gene IIA for cells that have integrated multiplecopies of the vector into their genomes).

pMMTC (FIG. 3) is a mammalian cell expression vector. The gene to beexpressed is cloned into XhoI and/or NotI sites so that the expressionof the gene is driven by a control region (MSV-mMT1) that comprises themouse sarcoma virus enhancer (MSV) and the mouse metallothionein gene 1promoter. The SV40 splicing region and the polyadenylation site serve toterminate the transcription and to ensure proper control ofpost-transcriptional events.

The bacterial neo gene is flanked upstream with the SV40 origin ofreplication and the SV40 early promoter and downstream by the SV40splicing region and the polyadenylation site. This neo expression unitconfers transfected mammalian cells resistance to geneticin (G418) andalso confers upon transformed E. coli. resistance to kanamycin.

The pBR322 origin of replication (Ori) serves as the origin forautonomous replication of the plasmid DNA in E. coli. The humanmetallothionein structural gene IIA, which confers resistance to heavymetal ions such as Cd²⁺ on mammalian cells, was used to select formammalian transfectants that have integrated multiple copies of theplasmid.

Construction of mammalian expression vector pMMTC

Plasmid pRSN (Low et al., JBC 266;19710-19716, 1991) was cut with therestriction enzyme BamHI and resolved by agarose gel electrophoresis. ADNA fragment of about 2685 bp was gel-purified. This BamHI fragmentcontains the expression unit for the E. coli neo gene in both mammaliancells and E. coli.

Plasmid pBPV (Pharmacia: product number 27-4390; see FIG. 4 and belowfor full description) was cut with restriction enzyme BamHI and thentreated with calf intestinal alkaline phosphatase (CIAP). After beingresolved by agarose gel electrophoresis, a BamHI fragment of about 4570bp was gel purified. This 4570 bp fragment contains the pBR322 origin ofreplication in E. coli, the Ampicillin (Amp) resistance gene and anexpression cassette composed of the mouse sarcoma virus enhancer and themouse metallothionein gene 1 promoter followed by a multiple cloningsite and the SV40 splicing junction and polyadenylation signal.

This 4570 bp fragment was ligated to the above 2685 bp BamHI fragment.The resulting plasmid was named pMMTN (FIG. 2). A plasmid phMT (Karin etal (1982): Nature 299, 797-802) was cut with HindIII and blunt-endedwith Klenow fragment of DNA polymerase I. A 3100 bp fragment, containingthe human metallothionein gene II A, was gel-purified. Plasmid pMMTN wascut with ScaI (which is within the Ampicillin resistance gene), treatedwith CIAP, and then ligated with the 3100 bp fragment obtained fromphMT.

The product of this ligation was transformed into E. coli. Selection wasperformed for kanamycin resistance (conferred by the neo gene) and Ampsensitivity (due to the insertion of the 3100 bp fragment into thestructural gene for Amp resistance). The final plasmid construct wasconfirmed by restriction enzyme digestion and was named pMMTC. pMMTC isabout 10350 bp in length.

GENEOLOGY: pBPV (12516 bp)

(Nucleotide numbers refer to numbering in the reference)

MSV enhancer (388 bp)

Dhar. R. et al, Proc, Natl. Acad Sci. U.S.A. 77, 3937 (1980). Nuc529-142

BamHI/BglII linker CCGGATCTG

5′-end of metallothionein promoter (295 bp) Nuc 1-295

3′-end of metallothionein promoter (368 bp) Glanville, N. et al Nature292 267 (1981). Nuc 300-68

Multiple cloning site and additional nucleotides from constructionCTCGAGCCGCGGCCGCTTCGAGG (SEQ ID NO:1)

SV40 small T-antigen splice (612 Patent Bulletin No.) andpolyadenylation (235 Patent Bulletin No.) signals

Buchman, A. R. et al DNA Tumor Viruses, Cold Spring Harbor Laboratory,pg 799 (1980), Nuc 4713-4102 and 2772-2538

BPV genome (7945 bp)

Chen E. Y. et al Nature 299,529 (1982). Nuc 4451-7945 and 1-4450

pML2: a derivative of pBR322 with a deletion between bases 1,095 and2,485 (2,632) bp)

(1) Balbas. P., et al Gene 50.3 (1986).

(2) Sarvor. N. et al Proc. Natl Acad. Sci U.S.A. 79,7147 (1982). Nuc376-1095, 2485-4363 and 1-33

BglII/BamHI linker GAGATCCGG

EXAMPLE 3

Insertion of human β-interferon expression DNA into pMMTC

The β-interferon coding sequence was retrieved from human genomic DNA byPCR with two oligonucleotides. The 5′ oligo(GGGGTACCATGACCAACAAGTGTCTCCTC, SEQ ID NO:2) was modified in such a waythat the sequence (CCACCATG) around the initiation ATG codon favoursefficient translational initiation (Kozak (1984): NAR 12,857-872). Thesequence of the 3′ oligo was GGAATTCTTCAGTTTCGGAGGTAACCTGT (SEQ IDNO:3). This modified expression sequence for β-interferon was insertedinto the XhoI and NotI sites of pMMTC. The insertion and correctorientation was confirmed by restriction mapping, PCR or sequencing. Theresulting plasmid was named pMMTC/IFNβ.

EXAMPLE 4

Establishment of CHO cell clones that constitutively secrete high levelsof functional human β-interferon

CHO cells were transfected with pMMTC/IFNβ as described (Low et al., J.Biol. Chem. 266;19710-19716, 1991). Cells were selected in G418 (800μg/ml) for 7-10 days to allow growth of stably transfected cells. Thecells were then incubated in medium with 50-100 μM Zn²⁻ ions for 24 to48 hr to induce the expression of human metallothionein and thenincubated in a medium with step-wise increasing concentration of Cd²⁺(final concentration 200 μM). Individual colonies was cloned andexpanded. The culture medium from the cloned cells accumulatedβ-interferon to a concentration of 10⁶ I.U./ml or more and 10⁶ I.U. ormore of β-interferon was secreted by 10⁶ cells in at most 24 hr.

EXAMPLE 5

Production of human β-interferon in CHO cells

Wild type Chinese hamster ovary cells CHO-K1 line (ATCC CCL-61) werepropagated in Dulbecco's Minimum Essential Medium (DMEM) containing 10%fetal calf serum. Cells were grown at 37° C. in an atmosphere of 5%carbon dioxide. These cells were transfected with the pMMTC/IFNβ plasmidto secrete constitutively high levels of functional human β-interferon.Cells were selected as described in Example 4.

During the selection with Cd²⁻, clones of transfected cells weremeasured for the antiviral activity of β-interferon to show that theywere constitutively secreting high levels of functional humanβ-interferon. Antiviral activity was measured according to the method ofArmstrong (Armstrong, Applied Microbiology 21, 723, 1971) modified byincluding 0.2 μg ml of actinomycin D in the viral challenge and reactingthe viral-induced C.P.E. directly. From these measurements, individualcolonies were isolated and expanded. Indeed several lines were found toproduce 10⁶ I.U./ml to 10⁷ I.U./ml of β-interferon when grown in plasticroller bottles.

One of these cell lines, GS38, was maintained in culture over 12 monthsto test its ability to maintain a consistent high level of β-interferonproduction. The GS38 cell line was maintained in plastic culture flasks(80 cm²) in DMEM containing lot fetal calf serum, 100 μg/ml penicillin,100 μg/ml streptomycin, 2.5 μg/ml amphotericin and 150 μM zinc sulphate(“regular medium”). The seeding of a roller bottle (1700 cm²) was doneby adding a culture flask (80 cm²) of GS38 cells into one 1700 cm²roller bottle and the cells were maintained in 200 ml of regular medium.

The medium from the roller bottle was discarded on day 2 and day 4 andreplenished with 200 ml of fresh regular medium each time. On day 6, theregular medium was discarded and the roller bottle was replenished with300 ml of serum-free DMEM medium which contained 2.5 mg/ml of humanserum albumin containing the list of additional ingredients listed inTable 1 (“serum-free medium”).

TABLE 1 Component Conc. Penicillin 100 μg/ml Streptomycin 100 μg/mlAmphotericin B 2.5 μg/ml ZnSO₄ 150 μM EX-CYTE (trade mark)* 1:1000Transferrin 2.5-5.0 μg/ml Insulin 5 μg/ml *EX-CYTE is an aqueous liquidsupplement from human serum sold by Bayer, Illinois, U.S.A..

On day 7, the serum-free medium was discarded and replenished withanother 300 ml of serum-free medium. On day 8, the serum-free medium wasagain discarded and replenished with another 300 ml of serum-freemedium. On day 9, the serum-free medium (300 ml) was harvested andreplenished by another 300 ml of serum-free medium. This harvestingprocedure was repeated daily for another 14 days.

From each roller bottle, a total of about 4.2 liters of GS38-producedβ-interferon (or GS38-IFNβ) was harvested. From 2.4×10⁶ to 3.6×10⁶ I.U.of β-interferon was obtained per ml of crude harvest from GS38 cells.This is equivalent to 1.35 mg to 2 mg of GS38-IFNβ per day from oneroller bottle of GS38 cells from about 5 mg to 6.7 mg per liter ofGS38-IFNβ from 1 liter of crude harvest per day. The crude GS38-IFNβ,when purified to homogeneity, had a specific activity of 5.37×10⁸I.U./mg of protein (bovine serum albumin), standardized to theGb23-902-531 standard (an NIH reference standard distributed by theNatl. Inst. Allergy and Infectious Diseases, NIH, U.S.A.).

The harvest of crude GS38-IFNβ was pooled and subjected to purificationby a combination of affinity and ion exchange column chromatographypurification (Tan et al, J. Biol. Chem. 254, 8067-8073, 1979; Edy et al,J. Biol. Chem. 252, 5934-5935, 1977; Knight et al PNAS U.S.A. 73,520-523, 1976). Pure GS38-IFNβ was obtained with about 70-80% recovery.The pure GS38-IFNβ when analysed was found to be homogenous according tothe following criteria of homogeneity:

A single molecular mass of an apparent molecular weight of 26,300 wasobserved on SDS-PAGE (15%) (FIG. 5a). This is similar to the molecularweight of a natural β-interferon produced by primary human diploidforeskin fibroblasts after the superinduction procedure of Tan et al(1970 and 1973) (see FIG. 5b). Note that the broad range molecularweight markers obtained from BIO-RAD were slightly different from theones used as previously reported by ourselves and others. The identityof these g-interferons (GS38-IFNβ and human fibroblast-producedβ-interferon) were verified by Western Blot (FIG. 5b) to belong to asingle average molecular mass of 26,300.

When subjected to hplc (Hewlett Packard 1090) C18 column chromatography,the protein peak of the material corresponded directly with theantiviral activity of interferon.

When subjected to amino acid sequencing, the material had the sequenceof β-interferon.

The amount of GS38-IFNβ produced by GS38 cells over 12 months was foundnot to change much. The cells produced from 2.35 to 3.6×10⁶ I.U./ml ofGS38-IFNβ throughout that period.

Five biological activities of GS38-IFNβ were assayed. The β-interferonfrom primary human fibroblasts referred to below was produced from earlyto mid-passage primary human foreskin fibroblasts according to thesuperinduction method of Tan et al (1970) with additional priming ofcells by 100 I.U. of β-interferon about 16 hours before superinduction.The resulting β-interferon was purified by affinity chromatography. Thefive activities which were assayed are:

1. Antiviral activity of β-interferon was assayed on either human MRC5fibroblasts or human hepatoblastoma cell line (HepG2) after the modifiedmethod of Armstrong (1971). Accordingly, the specific activity ofGS38-IFNβ was 5.37×10⁸ I.U./mg protein as assayed in MRC5 humanfibroblasts and referenced to the NIH β-interferon standard. Theantiviral activity of GS38-IFNβ in HepG2 cells was at least equal to or1.5 times more effective than natural β-interferon from human fibroblastcells. GS38-IFNβ is also about 2.2 times more effective than BETASERON(recombinant human β-interferon produced in E. coli) in protecting HepG2cells against a viral VSV challenge.

2. Cell growth inhibition assay of β-interferon (Tan, Nature 260,141-143, 1976) on the human hepatoblastoma cells as described forprimary human cells but applied to HepG2 cells was performed. However,the assay was performed in 2 cm² wells containing 1 ml of regular mediumand an initial HepG2 cell count of 3 to 5×10⁴ cells/well. Accordingly,GS38-IFNβ was as effective as natural primary human diploid fibroblastβ-interferon in inhibiting HepG2 cell growth as measured by this invitro assay.

3. Pharmacokinetics of subcutaneously injected β-interferon wasperformed in rabbits. Purified β-interferon from either GS38 or primaryhuman fibroblasts, or BETASERON from E. coli, was separatelyreconstituted in 4 mg of human serum albumin in 1 ml of phosphatebuffered saline (0.15M NaCl) pH 7.0 containing 20 mg trehalose. 0.7×10⁶or 1.5×10⁶ I.U. of an interferon was separately injected subcutaneouslyinto the back of albino rabbits of a weight of about 1.5 kg and about 2kg respectively.

Whole blood (500 μl) was withdrawn from the rabbits at 15 min., 30 min.,1 h, 2 h, 3 h, 4 h and 5 h. Serum from the drawn blood was then assayedfor the antiviral activity of β-interferon according to the modifiedmethod of Armstrong (1971). The results are presented in FIGS. 10 (a)and (b) showing that GS38-IFNβ has a higher bio-availability thanβ-interferon produced from primary human fibroblasts and BETASERON.

The maximum level of GS38-IFNβ (128-256 I.U./ml) occurred after 1 hour,and significant levels of GS38-IFNβ (64-128 I.U./ml) were found for atleast 5 hours in the serum of rabbits injected with 1.5×10⁶ I.U. ofGS38-IFNβ. This was unexpected. It is generally known that subcutaneousor intramuscular injection of human β-interferon results in no or lowserum levels of circulating human β-interferon.

4. Pharmacokinetics of a neat intravenous bolus of β-interferon inrabbits was also performed. 1 ml of each kind of β-interferon(GS38-IFNβ, natural interferon produced from primary human fibroblastsand E. coli BETASERON) containing approximately equal amounts ofβ-interferon (0.7×10⁶ I.U.) was injected into the rabbit ear vein. Blood(500 μl) was withdrawn at 5 min, 10 min, 15 min, 30 min, and 90 min. Theserum was assayed for the antiviral activity of β-interferon accordingto the modified method of Armstrong (1971). The result is shown in FIG.11 where the half-life (t½) of GS38-IFNβ is 13.6 min, compared toprimary human fibroblast-produced β-interferon (t½=4.4 min) or BETASERON(t½=3.8). According to standard methodology, the total amount ofβ-interferon injected was divided by the blood volume to estimate thestarting concentration of β-interferon at time zero. The blood volumewas assumed to be 5% of the body weight of the rabbit. The time to decayto 50% of this starting concentration was t½.

5. The dosage effect of injecting increasing amounts of GS38-IFNβ wasinvestigated. Rabbits of about 2 kg were injected subcutaneously withincreasing amounts of GS38-IFNβ, in particular with 1.2×10⁶ I.U.,2.5×10⁶ I.U. and 10.1×10⁶ I.U. of GS38-IFNβ. The results in FIG. 12 showthat increasing doses of GS38-IFNβ injected subcutaneouslyproportionally increase the measurable level of GS38-IFNβ in the serumof the injected rabbits.

EXAMPLE 6

Analysis of oligosaccharides associated with GS38-IFNβ

GS38-IFNβ is a glycoprotein. The oligosaccharides associated withGS38-IFNβ were quantitatively released and recovered. The N and O linkedglycans were released by treatment with anhydrous hydrazine. In thisprocedure, the protein backbone is converted into amino acid hydrazones.Intact reducing glycans are separated, recovered and labelledfluorimetrically with 2-aminobenzamide.

More specifically, a sample of GS38-IFNβ (1-2 mg) was subjected tovigorous sample preparation, involving lyophilisation (<50 millTorr, >24 hours), introduced to a GLYCO PREP 1000 (an automated systemfor release and recovery of glycans from glycoproteins, (Oxford GlycoSystems, GB) and the oligosaccharides were released and recovered usingthe “N+O” program. The sample was fluorescently labelled by reductiveamination with 2-aminobenzamide.

The sample was then applied to Whatman 3MM chromatography paper andsubjected to ascending paper chromatography using1-butanol/ethanol/water (4:1:1). The labelled sample remaining at theorigin was subsequently eluted with water. This procedure leads to thequantitative (and non-selective) recovery of the total pool ofoligosaccharides associated with the GS38-IFNβ sample as2-aminobenzamide labelled oligosaccharide.

The pool of labelled oligosaccharides was fractionated and analysed asfollows:

The labelled oligosaccharides were analysed for their chargedistribution by hplc anion exchange chromatography. Accordingly, analiquot of the total pool of 2-aminobenzamide-labelled oligosaccharideswas subjected to hplc anion exchange chromatography on a GLYCO SEP Ccolumn (Oxford GlycoSystems, GB) using acetonitrile and ammonium acetateas eluent. The labelled glycans eluted from the column were detectedusing the fluorometer at λex=356 mm λem=450mm. The resultantchromatogram is shown in FIG. 6.

It is shown from FIG. 6 that the oligosaccharides associated withGS38-IFNβ consist of both neutral and acidic components. To determinethe nature of the acidic substituents, an aliquot of the total pool offluorescently labelled oligosaccharides was incubated with neuraminidase(derived from Arthrobacter ureafaciens). An aliquot was again subjectedto GLYCO SEP C chromatography. The resultant chromatogram is shown inFIG. 7.

No acidic oligosaccharides were detectable after incubation with theneuraminidase. Hence, the oligosaccharides that carry an acidicsubstituent do so only because they possess a covalently linkednon-reducing terminal outer-arm sialic acid residue. The relative molarcontent of neutral and acidic oligosaccharides in the total pool wasdetermined by integration of the chromatographic peaks (FIG. 6). Theresults are as follows:

Neutral 10% ± 0.8% (to 1 s.d.) Acidic 90% ± 0.6% (to 1 s.d.) s.d. =standard deviation

2. Size-distribution of the total pool of deacidified oligosaccharidesreleased from GS38-IFNβ

An aliquot of the total pool of deacidified 2-aminobenzamide labelledoligosaccharides was subjected to high resolution gel permeationchromatography using the RAAM 2000 (Oxford Glyco Systems, GB). Theresulting gel permeation chromatogram is shown in FIG. 8. As a note ofexplanation, the fluorescently labelled deacidified oligosaccharideswere suspended in an aqueous solution of a partial acid hydrolysate ofdextran, and applied to a RAAM 2000 (eluent of water, maintained at 55°C., constant flow 80 μl/min over 10.6 hours). Detection was by anin-line fluorescence flow detector (to detect fluorescently labelledsample), and an in-line differential refractometer (to detect individualglucose oligomers).

Numerical superscripts in FIG. 8 represent the elution position, of thenon-fluorescent, co-applied, glucose oligomers in glucose units (gu), asdetected simultaneously by refractive index. The hydrodynamic volume ofindividual 2-amino-benzamide labelled oligosaccharides is measured interms of glucose units, as calculated by cubic spline interpolationbetween the two glucose oligomers immediately adjacent to thefluorescently labelled oligosaccharide.

It is clear that at least 6 discrete oligosaccharide are identifiablewithin the dextran calibration range, and their effective hydrodynamicvolumes are as follows:

20.7 gu 14.5% 17.6 gu 23.4% 14.5 gu 29.8% 12.2 gu 26.4% 11.1 gu  2.1% 1.0 gu  3.8%

Annotation of hydrodynamic volume is accurate to ±0.1 gu for all volumes≦20 gu. The conjugation of the glycans with 2-aminobenzamide (2-AB)decreases the hydrodynamic volume of the glycans by a constant value.The hydrodynamic volume of the 2-AB labelled glycans (λf) is calculatedfrom hydrodynamic volume of the unreduced glycans (λ) using thefollowing equation:

λf=1.2λ−1.96

3. Molecular weight distribution of the de-acidified glycans releasedfrom GS38-IFNβ

Since peaks were detected outside the dextran calibration range (FIG. 8)and particularly in the void volume, it was necessary to obtain amolecular weight distribution in order to establish what carbohydratespecies were present. An aliquot of the de-acidified glycan pool wasprepared on a matrix of 3.5-dihydroxybenzene. A Matrix-Assisted LaserDesorption Ionisation-Time of Flight (MALDI-TOF) mass spectrum wasobtained in positive ion mode (i.e. molecular ion plus sodium). Thefollowing ions could be assigned to carbohydrates (FIG. 9).

Molecular Ion Na 1929.9 2292.5 2660.1 3019.1

4. SUMMARY

The Glycoprotein GS38-IFNβ carries oligosaccharides with the followingstructural characteristics:

(i) Neutral (no acidic substituents): 10% ± 0.8% Acidic: 90% ± 0.6% (ii)The total desialylated oligosaccharide pool is heterogeneous, with atleast 6 distinct structural components present in the total pool. (iii)The MALDI-TOF mass spectrometry data and the RAAM 2000 data can besummarised as follows: Mass Composi- Calculated gu detected tion Massequivalent 1786.2 5Hex, 1782 11.1 4HexNAc 1 2AB, Na 1929.9 5Hex, 1dHex,1928 12.2 4HexNAc, 1 2AB, Na 2295.5 6Hex, 2293 14.5 1dHex, 5HexNAc 12AB, Na 2660.1 7Hex, 2658 17.6 1dHex, 6HexNAc 1 2AB, Na 3019.1 8Hex,3023 20.7 1dHex, 7HexNAc, 1 2AB, Na Hex = Hexose, dHex = deoxyHexose,HexNAc = N-Acetylhexosamine, 2AB = 2-aminobenzamide, Na = sodium ion.

NB. The peak which elutes at 1.0 gu will be included in the matrix inMALDI-TOF mass spectrum and is therefore not detected.

EXAMPLE 7

Expression of human erythropoietin in CHO cells using pMMTC

cDNA encoding human erythropoietin (EPO) was derived by PCR with pfupolymerase using human kidney mRNA that had been reverse transcribed.The nucleotide sequence of the 5′ and 3′ PCR primers are as follows:

5′PCR Primer:

5′GTGGATCCGCCGCCACC/ATG/GGG/GTG/CAC/GAA/TGT/CCT/GCC/TG-3′ (SEQ ID NO:4,the CCGCCGCCACC sequence (SEQ ID NO:5) before the ATG initiation Metcodon was designed for optimal translation of the resulting mRNA); and

3′PCR Primer:

5′-GATCTAGACAGTTCTTGTCAATGAGGTTGAAG-3′ (SEQ ID NO:6)

The PCR product was gel-purified, cut with restriction enzymes BamHI andXbaI, and then ligated into pGEM-11Z plasmid that has been cut withBamHI and XbaI. After confirming the nucleotide sequence of the EPOcoding region, the cDNA was retrieved from pGEM-11Z plasmid by cuttingwith XhoI and NotI. The EPO cDNA was gel-purified, and inserted into theXhoI-NotI sites of pMMTC, giving rise to a plasmid referred topMMTC/EPO.

Wild type CHO cells (CHO-K1) were transfected with pMMTC/EPO andinitially selected with G-418 for 7 days and then with graduallyincreased concentrations of Cadmium (4, 8, 16, 32, 64 and 92 μM). Abouta few thousand colonies were obtained after the initial G418 selection.When the Cadmium concentration was 64 μM, about 100 colonies remainedviable. Among these 100 colonies, 60 were individually isolated andexpanded, and assayed for the levels of EPO in their culture media byWestern blot, resulting in identification of 8 high expressing colonies(referred to as E15/1, E15/3, E15/8, E15/10, E15/13. E15/18, E15/26 andE15/30, respectively).

The remaining colonies were further selected in media with 92 μM Cadmiumand several colonies remained viable after this selection, from which 6colonies (termed C5, C10, C11, C12, C14 and C15, respectively) wereindividually isolated and assayed for the levels of EPO expression. Thelevels of EPO secretion by the 8 high expression colonies after 64 μMCadmium selection and the 6 colonies after 92 μM Cadmium selection werefurther compared by Western blot.

Cells of the selected colonies were seeded onto 35 mm (in diameter)culture dishes. Upon confluency, 1 ml of culture medium was added toeach of them and cultured for 24 hrs. The media were then harvested and10 μl of each, together with a control 50 ng of EPO (lane 15) wereresolved by SDS PAGE and analyzed by Western blot using the Amersham ECLdetection system. The Western blot B shown in FIG. 13.

7 23 base pairs nucleic acid double circular other nucleic acid /desc =“Multiple cloning site of plasmid pBPV” unknown 1 CTCGAGCCGC GGCCGCTTCGAGG 23 29 base pairs nucleic acid single linear other nucleic acid /desc= “PCR primer” unknown 2 GGGGTACCAT GACCAACAAG TGTCTCCTC 29 29 basepairs nucleic acid single linear other nucleic acid /desc = “PCR primer”unknown 3 GGAATTCTTC AGTTTCGGAG GTAACCTGT 29 43 base pairs nucleic acidsingle linear other nucleic acid /desc = “PCR primer” unknown 4GTGGATCCGC CGCCACCATG GGGGTGCACG AATGTCCTGC CTG 43 11 base pairs nucleicacid single linear other nucleic acid /desc = “PCR primer fragment”unknown 5 CCGCCGCCAC C 11 32 base pairs nucleic acid single linear othernucleic acid /desc = “PCR primer” unknown 6 GATCTAGACA GTTCTTGTCAATGAGGTTGA AG 32 16 base pairs nucleic acid double circular othernucleic acid /desc = “Fragment of multiple cloning site of plasmid pBPV”unknown 7 CTCGAGCCGC GGCCGC 16

What is claimed is:
 1. A purified human β-interferon having a specificactivity of 4.8×10⁸ to 6.4×10⁸ I.U. per mg equivalent of bovine serumalbumin protein, wherein 25 to 65% of the oligosaccharides of thecarbohydrate moiety of said human β-interferon are tri-antennaryoligosaccharides.
 2. The purified human β-interferon according to claim1 wherein when 1.5×10⁶ I.U. of the interferon is injected subcutaneouslyinto the back of a rabbit of about 2 kg: (a) ≧128 I.U./ml of theinterferon is detectable in the serum of the rabbit after 1 hour, and/or(b) ≧64 I.U./ml of the interferon is detectable in the serum of therabbit after 5 hours.
 3. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and, as an activeprinciple, the human β-interferon as defined in claim 1.